Title

Author

Date of Award

Degree Type

Degree Name

Department

Entomology

First Advisor

Richard H Shukle

Committee Chair

Richard H Shukle

Committee Member 1

Ming-Chen Shun

Committee Member 2

Brandon J. Schemerhorn

Committee Member 3

Jeffery J. Stuart

Abstract

Mayetiola destructor Say, the Hessian fly, is a gall midge and a member of the Dipteran family Cecidomyiidae. It is a common pest of wheat found throughout all of the major wheat growing areas of the world and poses a serious economic threat to the United States (US), particularly in the Southeast winter wheat region. Damage to wheat is done solely by feeding first and second in-star larvae. Hessian fly (Hf) infestations result in a loss in grain yield by the stunting and/or killing of seedling wheat plants in the winter and by causing breakage at the nodes of the plant in the spring. Feeding begins as the larvae settles at the base of the plant and establishes a feeding site by creating a layer nutritive tissue. Control of Hf in the US is primarily performed through avoidance by planting after the bulk emergence of the fly and through planting resistant wheat cultivars which contain a Hf-specific R gene. In Israel, Hf is found throughout the primary agricultural region but is not considered an economic threat. No cultural practices are used to control the insect, and Hf resistant wheat cultivars are not deployed in commercial agriculture. Native grasses and wild wheat progenitors that can serve as alternative hosts are readily available in non-cultivated areas. The sampling Hf in Israel will provide information from a Mediterranean population which is as close to Fertile Crescent, the center of origin to both Hf and the domestication of wheat, as can be currently sampled. This will allow the examination of population structure in Israel, of differential expression of effector proteins, of virulence to Hf R genes when resistant wheat cultivars are not deployed, and of the genetic inheritance of avirulence genes (Avr) in virulent Hf. This will allow advancement in the understanding of the Hf-wheat interaction that can be used to create more effective and long-lasting control of Hf in US. Samples of a dipteran pest of wheat from multiple locations in the agricultural area of Israel were tested to confirm identity, describe local populations and suggest the use of deploying resistance (R) genes in wheat cultivars for control of Hf. Morphological evaluation of adults and a free-choice oviposition preference test documenting that females overwhelmingly preferred to oviposit on wheat instead of barley supported the identification of the Israeli samples as Hf. Using the cytochrome c oxidase subunit I (coxI), the Barcoding Region, nine haplotypes were revealed. These results supported the identification of Hf as all nine haplotypes fell within a single clade that was significantly separated from other gall midge species including Mayetiola hordei. A greenhouse culture was established for one of the sampling locations, Magen, and it was evaluated for virulence to 19 different R genes. .Magen was significantly virulent to 11 of the 19 R genes tested, and complementation analysis documented that, for four of the R genes tested, the Israeli Hf shared loci for virulence with Hf from the US. Levels of Hf infestation at seven Israeli fields were at least at the 5-8% level, which historically has indicated a significant yield loss. Microsatellite genotyping of the five Hf collections from Israel revealed two mixed populations in Israel that are distinctly separate from the single population in Syria. Evidence is emerging that some proteins secreted by gall forming plant-parasites act as effectors responsible for systemic changes in the host plant, such as galling and nutrient tissue formation. A large number of secreted salivary gland proteins (SSGPs), the putative effectors responsible for the physiological changes elicited in susceptible seedling wheat by Hf larvae, have been documented. However, how the genes encoding these candidate effectors might respond under field conditions is unknown. Microarray analysis was performed to investigate variation in SSGP transcript abundance among field collections from different geographic regions (southeastern US, central US, and the Mediterranean). Results revealed significant variation in SSGP transcript abundance among the field collections studied. The field collections separated into three distinct groups that corresponded to the wheat classes grown in the different geographic regions as well as to recently described Hessian fly populations. These data support previous reports correlating Hessian fly population structure with micropopulation differences due to agro-ecosystem parameters such as cultivation of regionally adapted wheat varieties, deployment of resistance genes, and variation in climatic conditions. Hf larvae produce a large number of secreted salivary effector proteins involved in effector triggered immunity that elicit systemic changes in susceptible wheat as well as trigger the defense response in resistant wheat. One of the avirulence effectors responsible for the interaction between Hf larvae and resistance gene H13 in wheat has recently been cloned and characterized using Hf populations from the US. Within the US, virulence is a sex-linked, recessive trait and was shown to be associated with three independent insertions that resulted in a loss of expression of the avirulence gene. Genetic crosses testing for the inheritance of virulence to H13 in Hf from Israel revealed that it is controlled by a sex-linked, recessive trait at a single loci. Additionally, no complementation occurred between crosses of virulent US and virulent Israeli Hf, supporting the hypothesis that virulence resides at the same locus in both populations. However, no insertions were identified in the coding region nor upstream or downstream of the coding region. Further, no single nucleotide polymorphisms or frame shifts corresponding to virulence were identified. These data suggest the molecular basis of virulence in the Israeli population to resistance gene H13 in wheat is not the same as in the US. As the most effective form of Hf control employs the planting of resistant wheat cultivars containing one or more H genes, frequent Hf sampling is required to monitor the level of virulence present in locally adapted populations. A novel assay for detecting virulence in the field was created by sampling Hf males using sticky traps baited with Hf sex pheromone and the molecular marker for virulence to H13. The Hf gene that controls virulence in Hf to resistance gene H13 in wheat has recently been cloned and characterized, and diagnostic molecular markers for the alleles controlling avirulence and virulence are now available. Utilizing two separate PCR reactions, the six alleles for avirulence and virulence can be scored based on band size on a 2% agarose gel. The results support the most recent survey of virulence to H13 as scored through the testing of live insects infesting H13 wheat in the greenhouse. Throughout the southeast, all three avirulence alleles can be identified while the most frequently identified allele for virulence corresponded to a 5kb insertion into exon 1 of vH13. In South Carolina, the PCR assay is sensitive enough to detect the spread of virulence into two counties previously documented as 100% susceptible to H13.